Fig. 6

PIWIL1 may regulate MAPs through stabilizing mRNA, but not DNA methylation. a Localization of PIWIL1 in neurons. Dissociated cortical neurons were electroporated with Flag or GFP fused PIWIL1 plasmids (green). PIWIL1 was mainly localized to the cytoplasm, Scale bar, 20 μm. b Western blotting shows that PIWIL1 knockdown decreased the protein level of MAP1B, MAP2, and Tau, but not DCX. Treatment with the DNA methyltransferase inhibitor 5′AZA (2 μM) did not prevent the reduction of MAPs caused by PIWIL1 siRNA. c Neurons were electroporated with Scramble or RNAi plasmid. 48 h later, ActinomycinD (ActD) was added to inhibit transcription for 0, 3 or 6 h. MAP1B mRNA levels were measured by qPCR. The RNA levels at 0 h time point were set as 100 %. Knockdown of PIWIL1 resulted in a faster decay of MAP1B mRNA. Error bar, SEM. d Specific interaction between PIWIL1 and the mRNA of MAP1B. Upper panel, cortical neurons were transfected with Flag-fused HIWI or the vector (CMV-3xFlag). The RNA-protein complex was immunoprecipitated (IP) by anti-Flag antibody. The pull-down of HIWI was validated by Western Blotting, with actin as the indicator of equal input. Weak exposure image indicates that band of IgG group is the non-specific signal with lower molecular weight compared to HIWI-Flag. Lower panel, mRNA of MAP1B immunoprecipitated was revealed by semi-quantitative RT-PCR